H₂O₂ is the transferrable factor mediating flow-induced dilation in human coronary arterioles

Abstract

Rationale

Endothelial derived hydrogen peroxide (H₂O₂) is a necessary component of the pathway regulating flow-mediated dilation (FMD) in human coronary arterioles (HCA). However H₂O₂ has never been shown to be the endothelium-dependent transferrable hyperpolarization factor (EDHF) in response to shear stress.

Objective

We examined the hypothesis that H₂O₂ serves as the EDHF in HCA to shear stress.

Methods and Results

Two HCAs were cannulated in series (a donor intact vessel upstream and endothelium-denuded detector vessel downstream). Diameter changes to flow were examined in the absence and presence of PEG-Catalase (PEG-CAT).The open state probability of BK_Ca channels in smooth muscle cells (SMC) downstream from the perfusate from an endothelium-intact arteriole was examined by patch clamping. In some experiments a cyanogen bromide activated resin column bound with CAT was used to remove H₂O₂ from the donor vessel. When flow proceeds from donor to detector, both vessels dilate (donor: 68±7%; detector: 45±11%). With flow in the opposite direction, only the donor vessel dilates. PEG-CAT contacting only the detector vessel blocked FMD in that vessel (6 ±4%) but not in donor vessel (61 ±13%). Paxilline inhibited dilation of endothelium-denuded HCA to H₂O₂. Effluent from donor vessels elicited K⁺ channel opening in an iberiotoxin - or PEG-CAT sensitive fashion in cell-attached patches, but had little effect on channel opening on inside-out patches. Vasodilation of detector vessels was diminished when exposed to effluent from CAT-column.

Conclusions

Flow induced endothelial production of H₂O₂ which acts as the transferrable EDHF activating BK_Ca channels on the SMC.

Vasodilation induced by blood flow plays a pivotal role in the physiological control of vascular tone. The mechanism of flow-mediated dilation (FMD) is traditionally thought to involve the synthesis and release of vasodilators including nitric oxide (NO),^{1, 2} prostaglandins (PGI₂)³ and cytochrome P450 (CYP450) metabolites of arachidonic acid.⁴ Recent data from our laboratory indicate that FMD occurs in human coronary arterioles from patients with coronary disease via a novel mechanism requiring endothelial production of reactive oxygen species (ROS), specifically hydrogen peroxide(H₂O₂).^{5, 6} Several studies demonstrate that H₂O₂ elicits non-NO, non- PGI₂-induced smooth muscle hyperpolarization and relaxation in mouse mesenteric,⁷ human mesenteric,⁸ porcine coronary and human coronary arteries.^{9, 10} However, it is unclear whether H₂O₂ generated in the endothelium truly acts as an EDHF diffusing to the underlying vascular smooth muscle to elicit dilation, whether it acts locally within the endothelium to release a distinct vasodilating substance, or whether it acts via gap junctions to stimulate smooth muscle relaxation.^{11, 12} We hypothesized that H₂O₂ is indeed released from the endothelium and is the transferrable agent that diffuses to the smooth muscle to elicit hyperpolarization by opening K⁺ channels, with resultant vasodilation. We used a bioassay system, which combines videomicroscopic, patch clamping and histofluorescence techniques to test this hypothesis. This unique approach allows for direct determination of the specific role of H₂O₂ in FMD. We determined that FMD in human coronary arterioles requires endothelial release H₂O₂. H₂O₂ is the transferrable substance (EDHF) that activates Ca²⁺-activated K⁺ channels in the underlying smooth muscle, a process that requires intact intracellular components. This study provides the first direct evidence that H₂O₂ is the EDHF that mediates FMD in the human coronary microcirculation.^{13, 14}

METHODS

Videomcroscopic study

Atrial appendage tissue from human subjects undergoing cardiopulmonary bypass surgery was placed in oxygenated physiological salt solution (PSS) as described previously.⁵ Two human coronary arterioles (HCA, 250-300 μm) dissected from the endocardial surface of the atrial appendage were cannulated in tandem and equilibrated in separate chambers with an endothelium intact vessel upstream and an endothelium-denuded vessel downstream. Denudation was performed by injecting 2 ml of air through the lumen¹⁵ before cannulation. Diameters of donor and detector vessel were recorded after development of spontaneous myogenic tone with supplemental endothelin-1 (10^-10 to 5 ×10^-10 mol/L) given to achieve 30-50% reduction in passive diameter. Flow was produced by changing the heights of the reservoirs connected to the end of donor and detector arteriole in equal and opposite directions to generate a pressure gradient.⁴ Intraluminal diameter was measured at pressure gradients of 20 and 100 cm H₂O. To eliminate the effect of NO and PGI₂, N^ω-nitro-L-arginine methyl ester (L-NAME, 10^-4 mol/L) and indomethacin (10^-5 mol/L) were added to both vessel chambers throughout each experiment. In some studies, polyethylene glycol catalase (PEG-CAT, 500 U/ml) was first applied to the detector chamber 20 minutes before flow. Diameter change to flow was measured in both donor and detector vessels. After washing the chamber, PEG-CAT was applied to the lumen of the donor vessel and FMD was measured again 20-minutes later. The calculated percent dilation to flow was normalized to the maximal dilation in the presence of papaverine (10^-4 mol/L) added at the end of the experiment.

Vasodilation to direct application of H₂O₂ was examined in endothelium denuded HCAs in the absence and presence of paxilline (10^-7 mol/L).

In a separate study, a donor vessel was connected to a cyanogen bromide (CNBr) activated resin column bound with or without catalase (CAT) as described below. Effluent collected directly from the donor vessel (bypass column) or diverted through the column was applied to detector vessel incubated with 500 μl PSS in a separate chamber. Diameter changes of detector vessels were measured in response to addition of effluent in cumulative volumes of 30 μl, 300 μl, and 3000 μl from the donor artery or from column bound with or without CAT.

Patch clamp studies

HCA donor vessels with intact endothelium were cannulated in a vessel chamber with the outflow pipette connected to a 500-μl patch clamp chamber. The inflow reservoir was set at 100 cm H₂O. Coronary vascular smooth muscle cells (VSMC) acting as detectors, were enzymatically isolated from HCAs¹⁶ derived from the same atrial appendage as the donor HCA. Unitary K⁺ currents were measured in cell-attached or inside-out membrane patches of VSMCs bathed in PSS as described previously.¹⁶ The effect of vessel effluent on unitary K⁺ currents was evaluated by 2-min recording intervals while the cells were exposed to either PSS or vessel effluent. In some experiments, 100 nM iberiotoxin was added to the pipette solution to determine the role of large conductance Ca²⁺-activated K⁺ channels (BK_Ca). In some cases PEG-CAT was added to the bath solution. The open state probability (NPo) was determined by the event list analysis using criterion of 50% threshold crossing.

An expanded patch clamp method is available in the Online Data Supplement at http://circres.ahajournals.org.

Removing extracellular H₂O₂ released from donor vessel

We also conducted experiments to determine if the response to shear at the detector site was the result of donor endothelial. To determine if H₂O₂ was the substance transferred from the intact vessel and responsible for dilation of the detector vessel, we ran donor vessel effluent through a column containing beads of CNBr activated resin bound with CAT (10mg /ml, CAT-column) to remove extracellular H₂O₂. A column of beads with CAT free (CF-column) was used as a vehicle control. The effectiveness of CAT-column on removing H₂O₂ was confirmed by measuring H₂O₂ concentration in effluent collected from either column (CAT or CF) using the Amplex Red method (see below) and by detecting H₂O₂ in the detector vessel with fluorescence microscopy.

Quantitation of effluent H₂O₂

The presence of H₂O₂ in the effluent from the donor vessel or column (CAT or CF) was determined by quantitative measurement with an Amplex Red H₂O₂ assay kit. Effluent (50 μl) was added to a microplate well containing 100 μmol/L Amplex Red and 0.2 U/ml horseradish peroxidase. The plate was incubated for 30 min at room temperature while being protected from exposure to light. Absorbance was measured spectrophotometrically at 560 nm.

Fluorescence detection of H₂O₂

Five HCAs were isolated from the same tissue. One was used as a donor connecting to either CAT-column or CF-column. The others were subjected to endothelial denudation (detector vessels) and then incubated for 10 minute with Kreb’s solution, effluent from either the donor vessel or the CAT-column, or the CF-column, respectively. Dichlorodihydrofluorescein (DCFH, 5 μmol/L) was added in each light-protected chamber for 30 minutes to assess fluorescence intensity as described previously.⁵

Chemicals

All chemicals were purchased from Sigma. Fluorescence dyes and Amplex Red kits were obtained from Molecular Probes.

Statistics

All data are expressed as mean ± SEM. Percent dilation was calculated as the percent change from the pre-constricted diameter to the diameter after agonist or flow (maximal diameter was measured after papaverine; 10^-4 mol/L). Data from vessels or cells exposed to flow or vessel effluent before and after antagonist treatment were compared using a one way ANOVA with repeated measures for dose and condition. The relative fluorescence of cells exposed to bath solution or vessel effluent was compared using a one way ANOVA. All differences were judged to be significant at the level of P<0.05.

RESULTS

HCAs were obtained from 24 patients with a mean maximal passive internal diameter 277±16 μm. Patient demographics including diagnoses are summarized in Online Table I.

Presence of a diffusible vasodilator in endothelium of HCA

The bioassay system was used to confirm the transferable nature of the EDHF responsible for FMD in HCA. As illustrated in figure 1A, when flow proceeded from donor to detector, both vessels dilated (100 cm H₂O: donor: 68±7%; detector: 45±11%, n=5). As anticipated, when flow was reversed (figure 1B), only the donor (endothelium containing vessel) dilated (100 cm H₂O; donor 40±12%; detector -11±8%, n=5, P<0.05 vs donor) suggesting a transferable endothelial dilator is essential for FMD in HCAs.

Figure 1.

Two HCAs were connected in series with donor (with endothelium, +E) upstream and detector vessel (without endothelium, -E) downstream. A. When flow proceeds from donor to detector, both donor and detector vessel dilated. B. With flow was reversed, only the donor dilated, but not detector vessels.

Necessary role for H₂O₂ in FMD of HCA

When the detector vessel was treated with PEG-CAT (500 U/ml in chamber) for 20 minutes, FMD was prevented in the detector (100 cm H₂O: 6 ±4%) (figure 2B), but not donor (100 cm H₂O: 61 ±13%) (figure 2A). Perfusion of PEG-CAT through the lumen of both vessels, eliminated FMD in both vessels, (figure 2A and 2B) suggesting that H₂O₂ from the donor vessel was responsible for smooth muscle relaxation in response to flow.

Figure 2.

Effect of PEG-CAT (500 U/ml) on FMD in donor vessels (A) and detector vessels (B). When PEG-CAT was placed in detector vessel chamber (cross bar), FMD was inhibited in detector vessels, but was reserved in donor vessels. When PEG-CAT was placed in the perfusate traversing both vessels (gray bar), FMD was eliminated in both donor and detector vessels.

Role of large conductance Ca²⁺-activated K⁺ channel (BK_Ca) in H₂O₂ mediated dilation of HCA

To examine the mechanism of H₂O₂-induced dilation in HCA, graded doses of H₂O₂ were applied to endothelium-denuded HCAs. As illustrated in figure 3, H₂O₂ dose-dependently dilated HCAs (100 μM: 95±2%), which was significantly reduced by 10^-7 mol/L paxilline (45±6%, n=6, P<0.05 vs control) indicating a critical role for BK_Ca channel in H₂O₂ induced dilation.

Figure 3.

Effect of paxilline on H₂O₂ induce dilation in human coronary arterioles. H₂O₂ elicited dose-dependent vasodilation, which was significantly inhibited by 100 nM paxilline.

Effect of vessel effluent on large conductance Ca²⁺-activated K⁺ channel (BK_Ca) activity in human coronary smooth muscle cells

To further examine the potential for smooth muscle hyperpolarization by the transferrable dilator agent, a modified bioassay system was employed using a donor vessel with effluent superfusing detector coronary vascular smooth muscle cells freshly isolated from the same patient’s heart. As illustrated in the sample traces (figure 4A) and summary data (figure 4B), the open state probability (NPo) of K⁺ channels in cell-attached patches was markedly enhanced when coronary smooth muscle cells were superfused with vessel effluent (control vs effluent, 0.0045±0.002 vs 0.11±0.05, n=8, P<0.05 vs control). The augmentation of K⁺ channel activity was abolished by iberiotoxin, a specific blocker of BK_Ca channels (0.002±0.001, n=4) indicating the specific involvement of BK_Ca channels.

Figure 4.

Effect of effluent from donor vessel on K⁺ channel activity in cell-attached patches of coronary smooth muscle cells. As indicated in sample traces (A) and summarized data (B), effluent from donor vessel greatly enhanced open state probability in cell-attached patches of smooth muscle cells. The enhancement was blocked by 100 nmol/L iberiotoxin.

Role of H₂O₂ in vessel effluent induced activation of Ca²⁺-activated K⁺ channel

The role of H₂O₂ in mediating BK_Ca activity in response to vessel effluent was evaluated by PEG-CAT. Figure 5 shows that in the presence of PEG-CAT, BK_Ca activity-induced by vessel effluent was attenuated (NPo, without vs with PEG-CAT, 0.11±0.09 vs 0.005±0.004, n=4) consistent with H₂O₂ as the mediator of smooth muscle hyperpolarization. These results confirm a role for H₂O₂ in flow-induced activation of BK_Ca channels.

Figure 5.

Effect of PEG-CAT on K⁺ channel opening induced by effluent from donor vessel in cell-attached patches of coronary smooth muscle cells. The increased opening of K⁺ channels by effluent was abolished in the presence of PEG-CAT as illustrated in sample traces (A) and summarized data (B).

Effect of intracellular components on BK_Ca channel activity induced by vessel effluent

Hydrogen peroxide can open BK_Ca channels by a variety of direct and indirect mechanisms.^{17, 18} To determine whether the enhanced BK_Ca activity induced by vessel effluent is a direct effect of H₂O₂ or results from activation of other intracellular pathways, we performed inside-out patches where intracellular constituents were eliminated. In contrast to cell-attached patches, the NPo of BK_Ca channel activity was reduced when the patches were superfused with vessel effluent, (control vs effluent: 0.26±0.06 vs 0.089±0.04, n=6, P<0.05 vs control) (figure 6 A, B) suggesting activation of BK_Ca channels by vessel effluent requires the presence of intracellular molecules. CAT had no effect on BK_Ca channel activities in inside-out patches superfused with vessel effluent.

Figure 6.

Effect of effluent from donor vessel on K⁺ activity in inside-out patches of coronary smooth muscle cells. K⁺ channel activity was significantly reduced in response to vessel effluent after elimination of intracellular components. A. Sample traces. B. Data summarized from 6 experiments.

Effect of removing donor extracellular H₂O₂ on H₂O₂ production in detector vessels

Data presented thus far indicate that a transferrable EDHF mediates FMD in HCA and that H₂O₂ is critical in this process. To confirm that H₂O₂ is identical to that EDHF requires demonstration that removing it from the intercellular space between endothelial and smooth muscle cells eliminates downstream smooth muscle hyperpolarization. To test this, we utilized the bioassay coupled with a novel column containing cyanogen bromide beads covalently bound with catalytically active catalase. The column was placed in line between donor and detector vessels to remove hydrogen peroxide from the donor effluent. Efficacy of the method is shown in figure 7A, where flow generated 0.6±0.09 μmol/L H₂O₂ from donor HCA. H₂O₂ production was reduced when effluent was passed through the beaded columns (CF- and CAT-column). A greater reduction in H₂O₂ was observed in effluent from CAT-column (0.08±0.05 μmol/L, n=4, P<0.05 vs donor effluent Figure 7A). As seen in figure 7B and summarized in figure 7C, effluent from the donor vessel increased DCF fluorescence indicative of peroxide formation in detector HCAs (10±4, n=4, P<0.05 vs 1 ±0 when Krebs was used). Donor effluent through the CF-column did not affect the rise in fluorescence intensity in detector HCAs (6±2, n=4) but when the CAT-column was interposed a marked decrease in detector fluorescence was noted (0.7±0.3, n=4, P<0.05 vs CF-column). Thus CAT-bound beads but not naked beads effectively remove peroxides from the effluent of HCA exposed to flow.

Figure 7.

A. A significant reduction in H₂O₂ concentration was observed in effluent from CAT-column compared to effluent from CF-column. B. Sample images comparing fluorescence intensity of DCFH representing the production of H₂O₂. DCFH fluorescence intensities were increased in HCA exposed to effluent from donor vessel. Fluorescence intensities were greatly reduced in HCA incubated with effluent passing through the CAT-column. C. Summary of fluorescence intensities (normalized to control) in 4 arteries of each group. Donor effluent-induced increases in DCFH fluorescence were reduced in vessels exposed to effluent from CAT-column.

Effect of removing donor extracellular H₂O₂ on FMD of detector vessels

A marked reduction in dilation was observed in detector vessels incubated with donor effluent passed through a CAT-column (3000 μl effluent, 15.3±2.7%, n=4, P<0.05 vs CF-column) compared with effluent from a CF-column (3000 μl effluent, 31.6±2.1%), indicating the essential role of donor endothelial H₂O₂ in mediating dilation of detector vessels (figure 8).

Figure 8.

Comparison of dilator response of detector vessels to effluent from donor vessel, CF-column or CAT-column. Detector vessel dilated to effluent from donor vessels or CF-column in as volume of donor effluent increase. The dilator response was significantly reduced in detector vessels incubated in effluent from CAT-column.

DISCUSSION

EDHF plays a critical role in regulation of the human coronary microcirculation. The identity of the EDHF has remained elusive, although H₂O₂ has been shown to be an essential component of the signal transduction pathway for shear-induced dilation.^18-20 The present study employs a novel bioassay with videomicroscopic and patch clamp readouts, as well as a unique bioassay H₂O₂ quenching system to establish the role of H₂O₂ as the transferrable EDHF mediating FMD in HCA. The major findings of this study are two-fold: 1) endothelial H₂O₂ is the EDHF stimulated by shear stress that evokes hyperpolarization and relaxation in smooth muscle cells by a paracrine mechanism; and 2) H₂O₂-induced hyperpolarization and vasodilation result from the opening of BK_Ca channels in the smooth muscle membrane through an indirect effect requiring intact intracellular signaling.

Is H₂O₂ really an EDHF and is it responsible for FMD?

By definition, EDHF is a transferrable substance that relays a dilator signal from the endothelium to the underlying vascular smooth muscle. This transfer may occur via direct cell to cell communication as with potassium ions traversing gap junctions²¹ or by a paracrine mechanism involving endothelial production of the chemical which traverses the endothelial cell and acts on adjacent or regional smooth muscle cells (e.g. EETs²²). H₂O₂ has been proposed as an EDHF based on the observation that agonist- or flow- induced non-NO-non-PGI₂-mediated dilations are partially or completely prevented by catalase in several vascular beds from animals^{9, 10, 23} and humans.^{5, 8, 24} However, the conclusions derived from these studies are weakened by lack of direct evidence that endothelial H₂O₂ serves as the paracrine agent. Alternatively some other substance could be released from endothelial cell which traverses the extracellular space, stimulating release of H₂O₂ in the smooth muscle, or H₂O₂ could be acting within the endothelium to release a distinct EDHF. A final concern is that by studying intact vessels, it is not possible to eliminate direct communication between endothelium smooth muscle as being necessary for the dilator response involving H₂O₂. To circumvent these issues, the present study used a novel bioassay system to show that shear-induced endothelial derived H₂O₂ is the transferable factor responsible for smooth muscle hyperpolarization and vasodilation. This finding is supported by several lines of evidence. Firstly, H₂O₂ was detected from the effluent of donor vessels during flow. Second, FMD in both detector and donor HCAs was eliminated by application of PEG-CAT in into the vessel lumen. Finally, in detector HCAs, both the DCFH fluorescence intensity and dilator response were markedly diminished in vessels exposed to donor effluent in which H₂O₂ was removed by a resin column coated with CAT compared to vessels incubated with effluent from the CAT-free column. These results provide strong evidence that endothelial H₂O₂ is indeed the transferrable EDHF responsible for flow-induced vasodilation in HCAs, which is a unique feature of human coronary vascular response to flow, not observed in other species.

H₂O₂ activates BK_Ca channels

It has been reported that exogenous application of H₂O₂ hyperpolarizes smooth muscle cells in various vascular beds.^25-28 However, a variety of K⁺ channels contribute to this dilation depending on the species and vascular bed. In rat cerebral and porcine coronary arteries, BK_Ca channels mediate H₂O₂ -induced dilation since responses are blocked with tetraethylammonium²⁹ or iberiotoxin.^30,31 In canine coronary arterioles and rat mesenteric arteries, H₂O₂-mediated dilation involves activation of K_v channels.³² In piglet pial arteries, H₂O₂ induced dilation was prevented by glibenclamide, ²⁷ indicating the contribution of K_ATP channels.

H₂O₂ has been reported to have a bidirectional effects on BK_Ca channel activity.^{17, 33, 34} In porcine coronary smooth muscle cells H₂O₂ applied extracellularly increases opening probability.¹⁷ In contrast, when H₂O₂ is applied intracellularly in human embryonic kidney cells co-expressed with BK_Ca α- and β-subunits ^{35, 36} or in human umbilical vein endothelial cells³⁷, an inhibitory effect was observed. Oxidation of cysteine residue near the Ca²⁺ bowl sensor of BK_Ca α-subunit has been postulated as one of the mechanisms for the reduction of channel activity by H₂O₂.³³ This differential paracrine and autocrine effect of H₂O₂ on BK_Ca may be a factor contributing to the divergent results in different preparations. In addition, various concentrations of H₂O₂ were utilized in different studies ranging from 1 μM to 10 mM. A transition from proliferative to apoptotic signaling occurs across this range of concentration, which may also contribute to the conflicting results. The unique bioassay preparations used in this study provide a physiological (but unmeasured) concentration of H₂O₂ from a donor vessel and obviate many of the problems associated with pharmacological doses.

We found that activation of BK_Ca channels by H₂O₂ required the presence of intracellular constituents, since BK_Ca activity was not increased by H₂O₂ in inside-out patches. These results are consistent with the study by Barlow,³¹ where 300 μM H₂O₂ enhanced BK_Ca channel activity in cell attached, but not in inside-out patches. The mechanisms by which how H₂O₂ activates BK_Ca channel are complex and not well understood. Multiple signaling pathways have been implicated in H₂O₂-induced responses. The cGMP pathway plays prominently as H₂O₂ can activate guanylate cyclase directly, ^{38, 39} or oxidatively modify cysteine residues on PKG1α to produce dimerization and activation of this BK_Ca-opening kinase.⁴⁰ Other intermediate signaling pathways have been proposed as mechanisms for BK_Ca channel opening in response to H₂O₂, including arachidonic acid metabolites,^{28, 41, 42} protein kinase C,³⁸ and/or MAPK-ERK activation.¹¹

Study limitations

Several potential limitations warrant discussion. The amount of flow through each vessel was the same in both directions. However the luminal pressure in the vessel proximal to the higher reservoir may be greater than the luminal pressure in the more downstream vessel. It is possible that this difference in baseline of intraluminal pressure between donor and detector vessels influenced the dilator response to flow. To minimize this limitation, we used larger coronary arterioles (250-300 um) and pipettes with bigger tip sizes. Although a small difference in dilator response to flow was still observed in donor vessels with different direction of flow and a trend of lower FMD in detector vessels, this difference was not statistically significant and did not alter the interpretation of our data.

In patch clamp studies, the cells were superfused with effluent from a donor vessel containing higher Ca²⁺ and lower K⁺ than standard bath solutions for single channel recording. Superfusion of cells with effluent may cause two confounding influences: 1) reducing channel conductance; and 2) increasing the effect of Ca²⁺ on channel open probability. While this could explain the relatively low single channel conductance we observed, similar values for conductance of human coronary BK_Ca channels were observed in this study and previous single channel recordings using standard patch clamp bath solutions,16 alleviating concerns about alteration of channel conductance by vessel effluent. The enhanced NPo of BK_Ca channels by effluent in cell-attached patches was inhibited by CAT indicating that H₂O₂ rather than Ca²⁺ was the primary effluent-mediating factor.

IBTX applied in single channel recording has to be located in the pipette solution as it is only active on the outside of cell membranes. To obtain control traces, the pipette tip was first dipped into and filled with the regular pipette solution without IBTX and then back filled with pipette solution containing IBTX. This is the only approach that can be utilized for single channel recording experiments. This is not an ideal preparation, since post-IBTX control traces could be contaminated by IBTX diffusion to the cell surface. However this diffusion takes time, and we observed no difference between pre- and post-IBTX control ion currents.

Although the majority of channel openings triggered by the vessel effluent in patch clamp recordings were BK_Ca channels, other channels with different conductance were also observed. Catalase did not alter the activity of these channels (unpublished observations). These channels were activated by vessel effluent but contributed only minimally to the overall current. Thus other channel types might provide some contribution to the net response to shear, but they are not involved in the prominent catalase-sensitive portion of FMD.

The CAT-column for removing extracellular H₂O₂ contained CNBr. Despite extensive washing of the beads prior to use, no toxicity or interference with fluorescence was observed. Vasomotor response was not altered by interposition of naked beads between donor and detector vessels.

We observed a reduction in H₂O₂ in effluent collected from CF control column with both Amplex Red and DCF fluorescence methods. Two possible explanations for this reduction include: 1) CNBr non-specifically breaks down H₂O₂; and 2) degradation of H₂O₂ with time. Further investigation of the effect of CNBr on H₂O₂ is beyond the scope of this study but it is important to note that: 1) use of naked beads provides an important control for these possibilities, and 2) the remaining H₂O₂ in the effluent was sufficient to elicit vasodilation similar to the vessel superfused with effluent without running through column.Vasodilation was abolished by effluent collected from CAT-bound column indicating the specificity of CAT column.

Clinical Implications

H₂O₂ has been proposed as a key mediator of vasodilation to flow in the diseased human heart.This study provides new fundamental information for understanding the dilator pathway involving transfer of H₂O₂from endothelial to smooth muscle cells.

Summary

Flow dilates donor and detector vessel dependent upon donor endothelial H₂O₂. H₂O₂ activates BK_Ca channel in cell-attached, but not inside-out patches of human coronary smooth muscle cells. Donor endothelial H₂O₂ is critical for regulating coronary smooth muscle relaxation. These results suggest that H₂O₂ induced by shear stress is a true EDHF, that activates BK_Ca channel and induces vasodilation by a mechanism requires intracellular constitutes of smooth muscle cells.

Non-standard Abbreviations and Acronyms

BK_Ca

Ca²⁺-activated K⁺ channels

CF-column

Catalase free column

CNBr

Cyanogen bromide

DCFH

Dichlorodihydrofluorescein

EDHF

Endothelium-dependent hyperpolarization factor

FMD

Flow-mediated dilation

H₂O₂

Hydrogen peroxide

HCA

Human coronary arterioles

L-NAME

N^ω-nitro-L-arginine methyl ester

NO

Nitric oxide

NPo

Open state probability

PEG-CAT

Polyethylene glycol catalase

PGI₂

Prostaglandins

ROS

Reactive oxygen species

VSMC

Vascular smooth muscle cells